GENOMIC COPIES & CFU/mL - (Oct/26/2009 )

Hey Guyz....

I need some help in DNA extraction....

I'm in the process of evaluating the GC:CFU ratio for various Mycoplasma species....

The following are the questions of interest.

Q1. How to achieve the maximal DNA extraction from the highest dilution of bacterial culture (10^1, 10^2, 10^3...) with out any loss or destruction of DNA.......I'm not sure if QIAGEN column method would be help, I tried using QIAGEN COLUMN extraction, using carried DNA, but during PCR reactions, carrier DNA is interfering with that of amplification.....this is leading to erroneous results.......so I need advise / ideas in achieving the complete DNA extraction from highest dilution....

Q2. How exactly Q-PCR can help in determining the GC:CFU ratio......

Please let know your views and ideas in this issues...

Thanks

Microbes in action on Oct 26 2009, 11:03 AM said:

Can you please elaborate what do you mean when you say carrieed DNA? and i have observed normally that the hand extraction protocol of the Meniatis (Sambrook- Molecular Cloning) gives better results than Qiagen
As far as the GC:CFU ratio is concerned i don know for sure but by Q PCR you can surely find out the Copy number and i guess the external formulaas can be used to find out the ratio!!!
Best luck!!!

Pradeep Iyer on Oct 26 2009, 07:35 AM said:

Hey,

Carrier DNA is used to elute your DNA of interest from the Matrix of the column that is used for extraction. This is performed when you extract DNA from highest dilution of bacterial cultures. This is to avoid any instances of extracted DNA stuck to the Matrix / column.

Thanks for your interest.